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Antimicrobial Agents and Chemotherapy, April 2009, p. 1645-1647, Vol. 53, No. 4
0066-4804/09/$08.00+0     doi:10.1128/AAC.01530-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

In Vitro Activity of Isavuconazole against Aspergillus Species and Zygomycetes According to the Methodology of the European Committee on Antimicrobial Susceptibility Testing

Susanne Perkhofer,^* Veronika Lechner, and Cornelia Lass-Flörl

Department of Hygiene, Microbiology and Social Medicine, Division of Hygiene and Medical Microbiology, Innsbruck Medical University, Innsbruck, Austria

Received 17 November 2008/ Returned for modification 12 December 2008/ Accepted 8 January 2009

Top ABSTRACT INTRODUCTION REFERENCES

 
We evaluated the MICs of isavuconazole (ISAV) against 96 isolates of Aspergillus species and 36 zygomycetes according to the methodology of the European Committee on Antimicrobial Susceptibility Testing. In addition, the in vitro activity was obtained for hyphal inocula. ISAV exhibited good antifungal activity against the tested isolates with the exception of Aspergillus niger and Mucorales. The in vitro activity of ISAV was comparable to that of voriconazole aside from Mucorales.

Top ABSTRACT INTRODUCTION REFERENCES

 
Invasive fungal infections are an increasing cause of morbidity and mortality in immunocompromised populations (2, 18). Despite correct antifungal treatment, the mortality rate of patients with invasive fungal infections remains high (7, 12). Isavuconazole (ISAV) (formerly known as BAL4815) is a novel and promising broad-spectrum triazole (16) in late-stage clinical development for the treatment of invasive aspergillosis and candidiasis. ISAV has proven in vitro activity against Aspergillus spp. and Candida spp. (13). In an experimental neutropenic murine model of disseminated Aspergillus flavus infection, ISAV treatment resulted in high survival rates compared to control (20).

The aim of this study was to test the in vitro activity of ISAV against a wide range of non-Aspergillus fumigatus species and a subset of various zygomycetes. Susceptibility testing was performed according to the methods of the Antifungal Susceptibility Testing Subcommittee of the European Committee on Antimicrobial Susceptibility Testing (EUCAST) (17). In addition, the minimal fungicidal concentrations (MFCs) were determined, and for a subset of fungi, we evaluated the activity of ISAV against hyphae of the various fungi (10, 15).

All fungi were recovered at the Innsbruck Medical University over a period of 10 years (1996 to 2006). The isolates were obtained from various specimens such as blood, respiratory tract specimens, biopsy specimens, and specimens of other deep sites. In total, we evaluated the MICs of 132 clinically relevant fungi such as A. fumigatus (n = 32), Aspergillus flavus (n = 16), Aspergillus terreus (n = 35), Aspergillus niger (n = 13), Rhizomucor spp. (n = 9), Absidia spp. (n = 8), Rhizopus spp. (n = 7), Cunninghamella spp. (n = 3), and Mucor spp. (n = 9).

ISAV was kindly provided as reagent-grade powder from Basilea Pharmaceutica (Basel, Switzerland). The azoles voriconazole (VOR) and posaconazole served as controls.

MICs were determined by using the reference procedure of the Antifungal Susceptibility Testing Subcommittee of EUCAST for spore-forming molds (17). Briefly, testing was performed in flat-bottomed microdilution plates by using RPMI 1640 medium supplemented with 2% glucose and an inoculum size of 2 x 10⁵ to 5 x 10⁵ CFU/ml. MIC endpoints were determined visually at 48 h and defined as the lowest drug concentration that resulted in a reduction in growth of 100% compared with that of a drug-free-growth control well. A. fumigatus ATCC 204306 and A. flavus ATCC 204304 were included as control isolates. The MFCs were evaluated by the method of Espinel-Ingroff et al. (4, 5) and defined as the lowest drug concentration that resulted in 99% killing.

The MICs for hyphae of Aspergillus spp. and zygomycetes were tested by the method of Lass-Flörl et al. (10, 15). Briefly, 100 µl of conidial solutions was added onto 96-well plates (Costar, Vienna, Austria) and incubated at 30°C for 12 to 20 h to allow the formation of hyphae. The outgrowth of hyphal length (50 to 70 µm) was determined with an inverted microscope. Wells were than washed and refilled with 100 µl RPMI medium supplemented with 2% glucose, and the drugs were added and incubated at 37° for an additional 12 to 18 h; the endpoints were read at 100% inhibition. For comparison of hyphal MICs, the metabolic activities of drug-treated hyphae were determined by their ability to reduce the tetrazolium compound 3-(4,5-dimethyl-2-thiazol)-2,5-diphenyl-2H-tetrazolium bromide (MTT), as described elsewhere (11). All tests were run in duplicate and were repeated twice.

The antifungal activity is expressed as the MIC range and the MICs at which 90% and 50% of isolates were inhibited (MIC₉₀ and MIC₅₀, respectively). MFC data are expressed similarly.

Our study showed ISAV to be active against a wide range of Aspergillus conidia and hyphae and to demonstrate activity against A. terreus, an amphotericin B-resistant species (Tables 1 and 2). MIC data obtained for posaconazole, VOR, and ISAV via EUCAST methodology are in agreement with MICs evaluated by CLSI methods. The average geometric means of MICs for ISAV against A. fumigatus, A. flavus, A. terreus, and A. niger were 0.63, 0.76, 0.68, and 2.36, respectively. ISAV demonstrated potent in vitro activity against Aspergillus spp. in a study testing 118 clinical isolates of A. fumigatus, A. flavus, A. terreus, and A. niger according to the CLSI method for broth dilution antifungal susceptibility testing of filamentous fungi (19). Sixteen Aspergillus isolates resistant to itraconazole, caspofungin, or amphotericin B were included, and ISAV showed no cross-resistance. None of the tested isolates exhibited ISAV MICs of >2 µg/ml (19, 20). Human pharmacokinetic data show a plasma level of >1.7 mg/liter (20). In our study, ISAV also displays fungicidal activity and MFCs were within 2 dilutions of the MICs for the various aspergilli tested (Table 3) as found by others (6).

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TABLE 1. In vitro susceptibility of BAL4815 against molds according to EUCAST methodology

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TABLE 2. MIC data obtained for hyphae of Aspergillus spp. and zygomycetes

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TABLE 3. MFC data obtained for Aspergillus spp. and zygomycetes

Several A. niger strains tested showed higher MICs than did A. fumigatus. This was not observed in the study by Warn et al. (19) nor in that by Guinea et al. (6). In a collection of 42 A. niger isolates, three isolates were resistant to itraconazole (MIC, >8 µg/ml) (8).

Zygomycetes are known to be resistant to VOR and echinocandins in vitro and in vivo (1). In our study, ISAV presented limited antifungal effects against zygomycetes (Table 1). Similar data were observed by others using CLSI criteria (3, 14): in comparison with itraconazole, ravuconazole, and VOR, ISAV showed partial activity against Mucorales. Only 11% of Rhizomucor spp. and 28% of Rhizopus spp. showed a MIC of <2 µg/ml.

The onset of invasive fungal infection is associated with the appearance of hyphae. Consequently, an agent must be active against the hyphal form in order to be clinically effective (2). ISAV exerted strong activities against the hyphae of A. fumigatus, A. flavus, and A. terreus (Table 2). For amphotericin B, the MICs of hyphae were 2 to 3 dilutions higher than conidial MICs (9). Hyphal MICs of zygomycetes against ISAV were similar to those for conidia. For hyphae, comparison of the visually determined endpoints with the results of the MTT method revealed that 87% of the visually determined MICs corresponded to a 95% or greater reduction in metabolic activity.

In conclusion, ISAV demonstrated impressive antifungal activity against hyphae and conidia of Aspergillus spp. and EUCAST methodology resulted in MIC ranges similar to those obtained with the CLSI reference method.

 
* Corresponding author. Mailing address: Department of Hygiene, Microbiology and Social Medicine, Division of Hygiene and Medical Microbiology, Innsbruck Medical University, Fritz Pregl Str. 3, 6020 Innsbruck, Austria. Phone: 43 512 9003 70729. Fax: 43 512 9003 73700. E-mail: Susanne.Perkhofer{at}i-med.ac.at

Published ahead of print on 21 January 2009.

Top ABSTRACT INTRODUCTION REFERENCES

 

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Antimicrobial Agents and Chemotherapy, April 2009, p. 1645-1647, Vol. 53, No. 4
0066-4804/09/$08.00+0     doi:10.1128/AAC.01530-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

This article has been cited by other articles:

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